NMR Characterization of Bioreactor Fermentation

To confirm the formation of propionate and other SCCA in bioreactor fermentations, we conducted NMR spectrometry in addition to HPLC‐RI. NMR was performed as previously described (Huertas‐Díaz et al., 2023 (link)). Briefly, 200 μL of sodium phosphate buffer (150 mM, pH 7.5) containing 5 mM trimethylsilylpropanoic acid (TSP) as internal chemical shift reference and 30% D₂O was added to 400 μL of fermentate collected from the bioreactors. The mixture was transferred to a NMR tube (4″ Tubes 5 mm, Bruker Biospin), and NMR spectroscopy was performed using a Bruker Neo‐IVDR 600 NMR spectrometer, operating at ¹H frequency of 600.03 MHz and equipped with a 5 mm ¹H BBI probe (Bruker BioSpin). All ¹H spectra were referenced to the TSP signal at 0 ppm. Spectra were manually phase and baseline corrected using Topspin 4.09 (Bruker Biospin) and signals were assigned using Chenomx NMR Suite 8.6 (Chenomx Inc), and the Human Metabolome Database (Wishart et al., 2022 (link)). To confirm the identification, we spiked samples with 1 μL of succinate (500 mM).